ABSTRACT
Circulating tumor cells (CTCs) and their clusters are the drivers of metastasis, but their interactions with capillary beds are poorly understood. Using microfluidic models mimicking human capillary bifurcations, we observed cell size- and bifurcation-dependent shedding of nuclei-free fragments by patient CTCs, CTC-derived explant cells and numerous cancer cell lines. Shedding reduced cell sizes up to 61%, facilitating their transit through bifurcations. We demonstrated that shed fragments were a novel class of large extracellular vesicles (LEVs), whose proteome was associated with immune-related and signaling pathways. LEVs were internalized by endothelial and immune cells, disrupted endothelial barrier integrity and polarized monocytes into M2 tumor-promoting macrophages. Cumulatively, these findings suggest that CTCs shed LEVs in capillary beds that drive key processes involved in the formation of pre-metastatic niches.
ABSTRACT
RNA polymerase II (Pol II) small nuclear RNA (snRNA) promoters and type 3 Pol III promoters have highly similar structures; both contain an interchangeable enhancer and "proximal sequence element" (PSE), which recruits the SNAP complex (SNAPc). The main distinguishing feature is the presence, in the type 3 promoters only, of a TATA box, which determines Pol III specificity. To understand the mechanism by which the absence or presence of a TATA box results in specific Pol recruitment, we examined how SNAPc and general transcription factors required for Pol II or Pol III transcription of SNAPc-dependent genes (i.e., TATA-box-binding protein [TBP], TFIIB, and TFIIA for Pol II transcription and TBP and BRF2 for Pol III transcription) assemble to ensure specific Pol recruitment. TFIIB and BRF2 could each, in a mutually exclusive fashion, be recruited to SNAPc. In contrast, TBP-TFIIB and TBP-BRF2 complexes were not recruited unless a TATA box was present, which allowed selective and efficient recruitment of the TBP-BRF2 complex. Thus, TBP both prevented BRF2 recruitment to Pol II promoters and enhanced BRF2 recruitment to Pol III promoters. On Pol II promoters, TBP recruitment was separate from TFIIB recruitment and enhanced by TFIIA. Our results provide a model for specific Pol recruitment at SNAPc-dependent promoters.
Subject(s)
Promoter Regions, Genetic , RNA Polymerase III/metabolism , RNA Polymerase II/metabolism , RNA, Small Nuclear/genetics , RNA, Small Nuclear/metabolism , HEK293 Cells , Humans , Mutation , Protein Binding , Protein Domains , Protein Transport , TATA Box/genetics , TATA-Box Binding Protein/metabolism , Transcription Factor TFIIB/metabolism , Transcription Factors/metabolismABSTRACT
Initiation of gene transcription by RNA polymerase (Pol) III requires the activity of TFIIIB, a complex formed by Brf1 (or Brf2), TBP (TATA-binding protein), and Bdp1. TFIIIB is required for recruitment of Pol III and to promote the transition from a closed to an open Pol III pre-initiation complex, a process dependent on the activity of the Bdp1 subunit. Here, we present a crystal structure of a Brf2-TBP-Bdp1 complex bound to DNA at 2.7 Å resolution, integrated with single-molecule FRET analysis and in vitro biochemical assays. Our study provides a structural insight on how Bdp1 is assembled into TFIIIB complexes, reveals structural and functional similarities between Bdp1 and Pol II factors TFIIA and TFIIF, and unravels essential interactions with DNA and with the upstream factor SNAPc. Furthermore, our data support the idea of a concerted mechanism involving TFIIIB and RNA polymerase III subunits for the closed to open pre-initiation complex transition.Transcription initiation by RNA polymerase III requires TFIIIB, a complex formed by Brf1/Brf2, TBP and Bdp1. Here, the authors describe the crystal structure of a Brf2-TBP-Bdp1 complex bound to a DNA promoter and characterize the role of Bdp1 in TFIIIB assembly and pre-initiation complex formation.
Subject(s)
RNA Polymerase III/metabolism , Transcription Factor TFIIIB/metabolism , Transcription Initiation, Genetic , Amino Acid Sequence , Crystallography, X-Ray , DNA/chemistry , DNA/genetics , DNA/metabolism , Humans , Models, Molecular , Nucleic Acid Conformation , Promoter Regions, Genetic/genetics , Protein Binding , Protein Domains , Sequence Homology, Amino Acid , TATA-Box Binding Protein/chemistry , TATA-Box Binding Protein/genetics , TATA-Box Binding Protein/metabolism , Transcription Factor TFIIIB/chemistry , Transcription Factor TFIIIB/geneticsABSTRACT
TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer.
